Transgenic approaches to define the functional role of dual site phospholamban phosphorylation.
Phospholamban is a critical regulator of the sarcoplasmic reticulum Ca2+-ATPase activity and myocardial contractility. Phosphorylation of phospholamban occurs on both Ser16 and Thr17 during isoproterenol stimulation. To determine the physiological significance of dual site phospholamban phosphorylation, we generated transgenic models expressing either wild-type or the Ser16 --> Ala mutant phospholamban in the cardiac compartment of the phospholamban knockout mice. Transgenic lines with similar levels of mutant or wild-type phospholamban were studied in parallel. Langendorff perfusion indicated that the basal hyperdynamic cardiac function of the knockout mouse was reversed to the same extent by reinsertion of either wild-type or mutant phospholamban. However, isoproterenol stimulation was associated with much lower responses in the contractile parameters of mutant phospholamban compared with wild-type hearts. These attenuated responses were due to lack of phosphorylation of mutant phospholamban, assessed in 32P labeling perfusion experiments. The lack of phospholamban phosphorylation in vivo was not due to conversion of Ser16 to Ala, since the mutated phospholamban form could serve as substrate for the calcium-calmodulin-dependent protein kinase in vitro. These findings indicate that phosphorylation of Ser16 is a prerequisite for Thr17 phosphorylation in phospholamban, and prevention of phosphoserine formation results in attenuation of the beta-agonist stimulatory responses in the mammalian heart.
Luo
W
W
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0575, USA.
Chu
G
G
Sato
Y
Y
Zhou
Z
Z
Kadambi
V J
VJ
Kranias
E G
EG
D000818:Animals; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D008822:Mice, Transgenic; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism; D010761:Phosphorus Radioisotopes / Q000378:metabolism; D010766:Phosphorylation; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D010761:Phosphorus Radioisotopes; D000099211:Phospholamban